Our long-term goal is to find out how migrating axons respond to guidance cues and are guided to their targets. A functional nervous system requires neuronal connections to be made in a highly detailed and stereotypic pattern. This specificity depends on the selection of pathways by neuronal growth cones. We focus on the guidance of specific axons in the C. elegans model system. As in other organisms, including humans, migrating axons have different responses to extracellular guidance cues. We do not have a very good idea of the mechanisms that permit some axon to migrate towards a source of a guidance cue, while other migrate away. How axons change their responsiveness to cues while undergoing morphological changes, such as branching, turning, and synapse formation is also not well understood. Using genetic screens we have identified several molecules likely to regulate axon responses, and now propose to investigate the mechanisms by which they act: (i) We found that in response to UNC-6/netrin and SLT-1/slit guidance cues the axon guidance receptor UNC-40/DCC is asymmetrically localized and recruits proteins such as MIG-10, which can promote axon outgrowth. (ii) UNC-6-ligated UNC-40 is proposed to signal the asymmetric localization of the receptor. We have found mutations in UNC-6 and UNC-40 that may alter the ligated UNC-40 conformation and we are studying how these changes alter the localization of UNC- 40 and can cause outgrowth in different directions. (iii) We found UNC-80 affects axon receptor activity and we are exploring whether this molecules influences the asymmetric localization of receptors relative to the source of the guidance cues. (iv) We have shown that two molecules, CLEC- 38 and RPM-1, negatively regulate the UNC-40 and the UNC-5 and SAX-3 axon guidance receptors, respectively. These molecules also positively regulate presynaptic development, suggesting a link between these processes that could help coordinate their activities. (v) We find evidence that acetylcholine secreted by target neurons regulates axon guidance receptors by controlling CLEC-38 and RPM-1 activity. This signaling could provide a means through which the targets regulate the development of approaching axons. We hope to extend our studies of these different molecules to help elucidate mechanisms that promote specific axon responses to guidance cues, thereby gaining a better understanding of how the neural circuits that underlie nervous system function develop.